Felix Ekwe

Degree project for Master of Science (Two Years) inBiology

Degree course in Plant Physiology 45 hec2015

Department of Biological and Environmental SciencesUniversity of Gothenburg

Examiner:Håkan PleijelSupervisor: Göran Wallin

Effects of nitrogen and phosphorus onphotosynthesis and growth of silver

birch (Betula pendula Roth.) andsunflower (Helianthus annuus L.)

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EFFECTS OF NITROGEN AND PHOSPHORUS ON PHOTOSYNTHESIS

AND GROWTH OF SILVER BIRCH (Betula pendula Roth.) AND

SUNFLOWER (Helianthus annuus L.)

Ekwe Felix MSc. Thesis 45 hp, 2015 University of Gothenburg, Department of Biological and Environmental Sciences

ABSTRACT The availability of nitrogen (N) and phosphorus (P) for plants are severely restricted, which reduces photosynthesis and growth in most terrestrial ecosystems (Harpole et al. 2011). This thesis investigates how variations in relative availability of N and P influences growth and photosynthesis in silver birch (Betula pendula Roth.) and sunflower (Helianthus annuus L.). Experiments with different nutrient solutions and addition rates of nutrients were carried out on young silver birch and sunflower seedlings to obtain plants with different N and P status. Two different nutrient solutions were used, one including all essential mineral elements in optimal proportions (N + P) and a second with the same proportion but without phosphorus (N – P). Seedlings of both species were grown in growth chambers and exposed to different light intensities, Silver birch (60 μmolm-2 s1) and sunflower (665 μmolm-2 s1). Gas exchange measurements were performed on intact plants. Nitrogen and biomass in the leaves and the whole plant was analysed after harvest The was results compared between species and treatments (N+P-treated and N-P-treated). Nutrient application had a significant effect on the CO2 and light response curves in both species irrespective of their growth chamber differences. Seedlings supplied with nutrient solutions N + P had higher net photosynthetic rates compared to seedlings supplied with N - P. The growth rate was stimulated in response to increasing addition of nutrients. At very high levels of N + P treatment, growth and photosynthesis was decreased in sunflowers indicating supra optimal concentrations. The resulting relationship of growth and photosynthesis with N + P treatment varied between the plant species. Sunflower responded more strongly to N + P treatment compared to silver birch. They had higher rate of increase in growth attributes (dry mass, fresh mass, leaf area and nitrogen content) and photosynthetic attributes (maximum carboxylation rate (Vcmax), maximum electron transport rate (Jmax), quantum yield ( ) and maximum photosynthetic rate (Amax)), indicating that sunflower is more efficient in utilizing N + P than silver birch.

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TABLE OF CONTENT  

INTRODUCTION:

Research questions:

MATERIAL AND METHOD:

plant materials and growth conditions

Nutrients Preparation

Gas exchange measurements:

Harvesting and chemical analysis:

Statistics

RESULTS:

Plant growth:

Accumulated nitrogen content:

Leaf nitrogen per area LNA (g m-2) vs nutrient treatment

Leaf nitrogen per mass LNM (mg g-1) vs nutrient treatment

Above ground nitrogen content AGN (mg) vs total nitrogen added

Above ground dry mass AGDM (g) vs accumulated above ground nitrogen (g):

Leaf dry mass per area (LMA g m-2) vs leaf nitrogen per area (g m-2):

Leaf Thickness (mm) vs leaf nitrogen per area (gm-2):

Maximum rate of carboxylation (Vcmax) on leaf nitrogen per area basis:

Maximum rate of electron transport (Jmax) vs leaf nitrogen per area:

Quantum yield vs Leaf nitrogen per area (gm-2):

Mitochondria respiration (Rd) vs Leaf nitrogen/area:

Leaf Photosynthetic capacity (Amax) as a function of nutrient supply in mg:

DISCUSSION:

Accumulated Nitrogen per unit leaf area and mass basis:

Change in Biomass:

Vcmax and Jmax in leaf N per unit area basis:

Quantum yield and Mitochondria respiration (Rd)on leaf N per unit area basis:

Leaf photosynthetic capacity (Amax) vs nutrient supply mg:CONCLUSION: ......................................................................................................................... 29

ACKNOWLEDGMENT:..……………………………………………………………………………….30

References: .............................................................................................................................. 30

 

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ABBRIVIATIONS

*: CO2 compensation point in absence of mitochondrial respiration

: Curvature of the light response curve

A/Ci: Leaf photosynthesis in relation to intercellular CO2 concentration

A/Qp Leaf photosynthesis in relation to photosynthetically active radiation

AGDM: Above ground dry mass

AGN: Nitrogen content of above ground plant parts

Amax: Maximum photosynthetsis at ambient CO2 concentration and light saturation

ANOVA: Analysis of variance

ATP: Adenosine triphosphate

G3P: Glyceraldehyde 3-phosphate

IRGA: Infra-red-gas-analyzer

Jmax: Maximum rate of photosynthetic electron transport

Kc: Michaelis-Menten coefficient of the activity of Rubisco with respect to CO2

Ko: Michaelis-Menten coefficient of the activity of Rubisco with respect to O2

LDM: Leaf dry mass

LFM: Leaf fresh mass

LMA Leaf mass per area

LNA: Leaf nitrogen per unit area

LNM: Leaf nitrogen per unit mass

LT: Leaf thickness (mm)

N: Nitrogen

N + P: Nutrient solution containing phosphorus

NADPH: Nicotine Amide Adenine Dinucleotide Phosphate

N - P: Nutrient solution without phosphorus

P: Phosphorus

PGA: 3-phosphoglycerate

R2: Squared error term

Rd: Mitochondria respiration

RuBisCO: Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

RuBP: Ribulose-1,5-bisphosphate

TPU: Trios phosphate use

Vcmax: Maximum carboxylation rate

: Quantum yield

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INTRODUCTION

Nitrogen (N) and Phosphorus (P) are mineral nutrients often limiting plant growth because they are required in large amounts in relation to their availability in soil (Harpole et al. 2011). A great deal of attention therefore has been paid to the cost and benefits of acquisition and use of these minerals at leaf and whole plant levels (Ingestad et al. 1995, Zubilaga et al. 2002). Leaf photosynthetic capacity (Amax) (maximum rate of carbon assimilation at light saturation and ambient CO2 level) has been reported to be highly correlated with leaf N and P content (Ingestad et al. 1995). Basically because the photosynthesis in plants with a C3 metabolism are regulated by the N-rich Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) and the chlorophyll/protein complexes which are functioning as the primary system for CO2 fixation and light harvesting, respectivley. (Field and Mooney 1986, Jiang et al 1993, Zubillaga et al 2002, Wang et al. 2012). P is a component of plants DNA, lipids and energy metabolites such as ATP and NADPH (Reich et al. 2009). The rich energy compounds (ATP and NADPH) produced in the light reaction of photosynthesis provides the energy used in the Calvin cycle to phosphorylate and reduce 3-phosphoglycerate (PGA) to Glyceraldehyde 3-phosphate (G3P) and regenerate Ribulose 1,5-bisphosphate (RuBP) (Farguhar et al. 1980). Phosphorus limitation therefore may inhibit or reduce the rate of photosynthesis by depressing the Calvin cycle activity, particularly the regeneration of ribulose-1,5-bisphosphate (RuBP). A fundamental relationship therefore exist between supply of nitrogen and phophorus and leaf photosynthetic capacity (Amax) (Field and Mooney 1986) irrespective of plant species or growth conditions. Although the importance of N and P to plant growth and photosynthesis is widely documented, its worthy to note that plants require these elements in adequate amounts. As reported by (Ingestad and Lund 1979, Knecht and Göransson 2004) plants require a certain minimum nutrient concentration to grow, and when the nutrients concentration is increased to optimum levels, it will increase growth rate relatively to the addition rate. Further increase of the nutrients concentration above optimum will not increase growth, and at very high concentrations the nutrient will become toxic, growth and photosynthetic rates will decrease and plants may die. It is thus important and essential when fertilizing the soil to supply nutrients wisely and adequately in order to avoid excessive application and to keep nutrient concentration around the optimal range.

The present study aims to determine the role of N and P on growth and photosynthesis in two plant species with C3 metabolism, silver birch (Betula pendula Roth.) and sunflower (Helianthus annuus L.). The nutrient concentrations or contents required to suport maximum growth and photosynthesis in silver birch and sunflower plants will be determined. Determining the effect of N and P on growth and photosynthesis in silver birch and sunflower may provide a clearer understanding on the central role of the elements in these species and will help to predict the optimal N and P fertilization requirement and to improve the practices of silver birch and sunflower production. Many attempts to determine the minimum and maximum external requirements of these nutrients for optimal growth and phosynthesis of these species have been made, e.g. (Ingestad and Lund. 1979, Ingestad 1981, Ericcson and Ingestad 1988, Knecht and Göransson. 2004). As noted by Ingestad (1981) the relative

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efficiency of N and P in photosynthesis and growth is directly dependent upon the sufficient avialability of all other elements. Therefore all elements were supplied in sufficient concentration using (Ingestad 1981, Hui-jun and Ingestad 1984) nutrient proportion scheme (Table 1). Phosphorus (P), as repoted by (Reich et al. 2009), influences the partitioning of nitrogen in plants. It is plausible therefore that the uptake and use efficiencies of one nutrient may be affected by the interaction with the other. The present study aims to verify the influence of P to N uptake and use efficiency in silver birch and sunflower.

In order to meet the objectives of this study, experiments were setup in the lab, in which different levels of N and P were relatively supplied to the plants, while keeping all other nutrients at low but non-limiting levels. All elements in solution were added in proportion to N according to (Ingestad 1981, Hui-jun and Ingestad 1984) nutrient proportion (table.1). The nutrients were supplied to plants using relative addition rate technique, which involved the addition of nutrients in relation to plant growth rate. By using this technique it was possible to supply the plants with their exact nutrient demands as they increased in size. Photosynthetic parameters such as maximum rate of RuBisCo activity (Vcmax), maximum rate of electron transport (Jmax), mitochondria respiration (Rd) and quantum yield (Φ) were determined and their relationship with N and P application verified and compared between species. The leaf properties such as leaf fresh mass, dry mass, area and thickness as well as above ground dry mass and nitrogen contents of the species were also determined and compared between species.

Research questions

To relate N and P to growth and photosynthesis of these plant species the following questions were addressed:

i. Is there a positive correlation between the addition of P and photosynthetic capacity? ii. Does P addition influence the relationship of Amax to variation in leaf N content?

iii. Does P fertilization affect the nitrogen use and uptake efficiencies of the two species? iv. Is there a difference between the two investigated species in their photosynthetic

responses to N and P supply? v. Are leaf properties (leaf dry mass, leaf mass per area and leaf thickness) influenced by

N and P supply and how are they related to photosynthesis? vi. What are the levels of N and P required by the species for maximum growth and

photosynthesis?

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MATERIAL AND METHOD

Plant materials and growth conditions

Seedlings of silver birch (Betula pendula Roth.) and sunflower (Helianthus annuus L.) were both supplied with different levels of N and P. Both experiments were carried out at Department of Biological and Environmental Science, University of Gothenburg. The study of silver birch was conducted in a growth chamber (PGW-36, Conviron, Canada). The Chamber temperature was set at 20°C, 18 hours daylight and 6 hours darkness. Relative humidity was 63% and the a photosynthetic photon flux density (i.e. 400-700 nm, PPFD) of 60 μmol m-2 s-1 measured using quantum meter with separate sensors (Apogee Instrument Inc. Model MQ-200). The sunflower experiment was conducted in a growth chamber (CLF plant climatics, AR-82L/3, Germany). Chamber temperature was 22°C day and 18°C night, RH was 75% and a PPFD of 665 μmol m-2 s-1 measured with quantum meter with separate sensors (Apogee Instrument Inc. Model MQ-200)

Nutrient treatment

Seedlings were treated with nutrients containing phosphorus (N + P) and without phosphorus (N – P). All mineral elements were added in proportion to nitrogen (N) according to (Ingestad. 1981, Hui-jun and Ingestad 1984) nutrient proportion (Table 1). The initial amount of nutrients added was made in relation to the biomass at the start. Plants with different nitrogen status were obtained by increasing the addition in various rates. Seedlings of both species were fertilised for a 6 weeks period before measurements of photosynthesis took place. The nutrient treatments of the silver birch and the sunflower started 8th Apr 2013 and 5th Sep 2013, respectively. Both experiments included two P treatments (N+P and N-P) plus three addition rates for silver birch (denoted 0%N, 1%N and 5%N) and five addition rates for sunflower (denoted 1%N, 2%N, 3%N 5%N, 7%N, 10%N) for each P treatment (Table 2). Table 1, Optimal nutrient (element) proportions in silver birch according to Ingestad (1981) and, Hui-jun and

Ingestad (1984). In this study, the same proportions were used for sunflower.

Elements Proportions in % of N

N 100 K 65 P 13 Ca 7 Mg 8.5 S 9 Fe 0.7 Mn 0.4 B 0.2 Zn 0.06 Cu 0.03 Mo 0.007

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Table 2. Treatments and replication in the experiment with the silver birch and sunflower. Addition rates are given as daily percentage increase in nutrient supply to match the increasing demand.

Gas exchange measurements

The gas exchange measurements of silver birch and sunflower leaves were conducted between 7th to10th May 2013 and 8th to 16th Oct 2013, respectively. The top most recent fully expended leaves were chosen. Measurements were done using a Li-Cor 6400 portable photosynthetic system (Open 6.1, LI-6400, Li-Cor Inc, Lincoln, NE, USA) equipped with an artificial light source (Figure 1). Leaf chamber temperature was maintained at 25oC and water vapor pressure deficit in the leaf chamber was maintained at level of 0.6-2.5 kPa, relative humidity at 40-50%. For both birch and sunflower, the leaves on which gas exchange measurement were conducted were harvested just after the gas analysis, their thickness was measured using an electronic digital Caliper. The leaf area (LA, m2 m-2) was measured with Win Folia (Win Folia, V 5.1a, Regent Inc, Quebec Canada). The fresh and dry mass was measured using a gram scale (Sartorius).

Species Addition rates in % per day

Plants treated with N + P solution

Plants treated with N - P solution

Silver birch 0 3 3

1 3 3

5 3 3

Sunflower 1 4 4

2 4 4

3 4 4

5 4 4

7 4 4

10 4 4

Figure 1. Using a Li-Cor 6400

portable photosynthetic system to take gas exchange measurements on sunflowers inside the growth chamber.

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The response of leaf photosynthesis (A) to intercellular CO2 concentration (Ci) and photosynthetic active radiation (Qp) were measured to establish A/Ci and A/Qp relationships. For silver birch A/Ci response measurements, the PPFD was maintained at 1500 μmol m-2 s-1

(PFD 400-700 nm) and reference CO2 levels used were 400, 60,125, 250, 400, 800, 1500, 2000, 400 μmol mol-1, and for sunflower CO2 reference levels were 60, 125, 200, 300, 400, 800, 1500, 2000, 400 μmol mol-1. After the A/Ci measurements the light response (A/Qp) was measured on the same leaf. Before taking the A/Qp measurements the chamber lamp use switched off for approximately 3 minute before running A/Qp measurements (to give time for the leaf to acclimate to darkness), CO2 concentration was fixed at 400 μmol mol-1. PPFDs of 0, 10, 20, 30, 50, 75, 100, 200, 600, 1200, 1500 μmol m-2 s-1 were use in both birch and sunflower light response measurements.

The data collected from A/Ci and A/Qp measurements was used to construct A/Ci and A/Qp

curves respectively. According to Farquhar et al (1980) and Sharkey (2007) photosynthesis is limited by one of three processes: The amount, activity and kinetics of RuBisCo, the rate of ribulose 1, 5-bisphosphate regeneration supported by electron transport, and trios phosphate use (TPU). Therefore values of the photosynthetic parameters; maximum carboxylation rate (Vcmax), electron transport rate (J), trios phosphate use (TPU) and dark respiration (Rd) were determined from the A/Ci curves constructed by fitting the A/Ci data into the (Farquhar et al. 1980, Sharkey. 2007) A/Ci model for C3-leaves (Figure 2).

The model by Farquhar et al (1980) and Sharkey (2007) for C3-leaves constrains intercellular CO2 (Ci) values < 100 ppm to be RuBisCo limited, hence Vcmax and Rd were estimated at low regions of the curve. The software to analyse the curves was set to always include a Ci of 100 ppm or less in the Vcmax calculation. When the rate of photosynthesis is RuBisCo limited (Farquhar et al 1980, Sharkey 2007) the response of photosynthesis to intercellular CO2 is described by the following equation:

-10

0

10

20

30

40

50

0 1000 2000 3000

A(µ

mo

lm-2

s-1 )

Ci (µmol mol‐1)

A/ci curve

Aobs Rubisco RuBP_regenFigure 2. Depicts the Farquhar et al (1980) and Sharkey (2007), photo-synthetic model. Photosynthetic rates observed (Aobs) derived from a gas exchange measurements at various intercellular CO2 concentrations (Ci) conducted at a leaf temperature of 25 °C were run in the model. The model constrained intercellular CO2 (Ci) values < 100 ppm to be RuBisCo limited and above >500 ppm to be limited by electron transport (RuBP regeneration).

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∗

∁ (1)

where A is rate of CO2 assimilation, Vcmax is maximum velocity of RuBisCo for carboxylation, Cc is CO2 partial pressure in the chloroplast, Kc is Michaelis constant for carbon dioxide, O = partial pressure of oxygen in the chloroplast, Ko is Michaelis constant for oxygen. Rd = Mitochondria respiration and Γ∗ is CO2 compensation point.

Jmax was estimated at higher Ci values of the curve >500ppm where photosynthesis is assumed to be limited by regeneration of RuBP via electron transport chain (Sharkey 2007, Farquhar et al 1980). Jmax was calculated from J using the (Medlyn et al. 2002) equation:

θJ2 - (αQ+ Jmax) J +αQJmax = 0 (2) Where J is the rate of electron transport, Q is the absorbed photon flux density, α (0.3) is the Quantum yield, θ (0.9) is the curvature of light response curve (Medlyn et al. 2002). The light curves (A/Qp) were used to estimate the photosynthetic quantum yield ( using the following equation:

(3)

Harvesting and chemical analysis

Harvesting of the silver birch and sunflower were done on the 20th May 2013 and 23th Oct 2013, respectively. For both species, the individual plants were separated into leaves, stems and roots, the roots were well washed with deionized water to get rid of all the vermiculite. The various parts of the harvested plants were weighed to get fresh mass (FM), then oven-dried at 70°C for 2 days and weighed to get dry mass (DM). Thereafter the individual plant parts were grind to a very fine powder using a grinding machine (Retsch model nr, MM301, Retsch Ltd, Germany). Nitrogen contents were determined by combustion of the powdered samples in an elementary analyser (Fison, model nr, AE 1108, CHNS-O, Italy). Results were compared between treatments as well as between the species.

Statistics 

A two way analysis of variance (ANOVA) with replication and a confidence level 0.05 were performed in Microsoft excel to test for the two factors P supply and nutrient addition rate on the collected data to provide a statistical comparison between the treatment means within the experiments.

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RESULTS

Plant growth

Fertilization was observed to have a prominent effect on the growth of both silver birch and sunflower. All plants began to show signs of nutrient deficiency (pale green) leaves during the initial phase of the experiment when no nutrient were added. However with the commencement of nutrient addition the paleness of leaves slowly began to disappear at all levels of nutrient supply except at 0%N birch plants and to a small extent at 1%N sunflower plants were the pale green leaf symptoms persisted. The rest of the fertilized plants in both species produced green, fresh and healthy leaves during the fertilization phase until harvest. In both experiments plants were observed to grow in accordance with the level of nutrient treatment, thus plants that received higher percentage levels of nutrients were observe to grow faster and vice versa. The roots of 0%N birch plants and of 1%N sunflower plants after harvest were observed to be larger with less branching compared to the rest.

Figure 3. Relationship between leaf nitrogen per unit area (LNA g m-2) vs nutrient treatment in % concentration per day (g) ((A (silver birch) and B (sunflower)), and leaf nitrogen per unit mass (LNM mg g-1) Vs nutrient treatment (mg) ((C (silver birch) and (sunflower)), P-values for A (N p=<0.0001, P p=0.57, N*P interaction p=0.25), B (N p=<0.0001, P p=<0.0001, N*P interaction p=0.13), C (N p=<0.0001, P p=0.02, N*P interaction p=0.29), and D, (N p=0.001, P p=0.02, N*P interaction p=0.58). +P and –P stands for plus phosphorus treated and without phosphorus treated respectively. Error bars are standard deviations from the mean.

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.

Figure 4. Relationship between nitrogen content of above-ground plant parts (AGN mg) vs total nitrogen added (mg). A, in silver birch (N p=<0.0001, P p=0.14, N*P interaction p=0.01), and B, in sunflower (N p=<0.0001, P p=0.04, N*P interaction p=0.06). +P and –P stands for plus phosphorus and without phosphorus respectively. Error bars are standard deviations from the mean.

Accumulated nitrogen content

A significant (p < 0.05) difference between treatments was found on accumulated leaf nitrogen content and accumulated above ground nitrogen content. By increasing the nutrient addition rates the nitrogen contents of leaves (Figure 3) and in the above ground part (Figure 4) increased. The highest nitrogen content was obtained at nutrient treatment levels of 5%N for silver birch and 10%N for sunflower. Phosphorus application was shown to affected leaf and above ground nitrogen content significantly (p < 0.05). Analysis of variance (ANOVA) showed that for both species the increase in leaf nitrogen per unit area (LNA), leaf nitrogen per unit mass (LNM) and above ground nitrogen (AGN) were all significantly (p < 0.05) affected by the nitrogen fertilization.

Leaf nitrogen per unit leaf area LNA (g m-2) vs nutrient treatment

Leaf nitrogen per unit leaf area was observed to increase as treatment levels increased in both species and in both N + P and N - P treated plants (Figure 3A, B). The increase was statistically tested to be significant (p<0.05) with increasing nitrogen application in both P treatments and species.

Silver birch (Figure 3A). There was only small difference in LNA between N + P and N - P treated plants at 0%N level. In the treatments with 0%N to 1%N level the N - P plants showed an increase in LNA of 0.33 g m-2 which was larger than in the N + P plants (0.18 g m-2). In the 1%N to 5%N, the N - P plants increased by (0.14 g m-2), which was less than in the N + P plants (0.35 g m-2), hence at treatment levels of 5%N, the rate of increase in LNA in plants treated with N + P was more than twice higher than those treated with N - P. Therefore although the N - P treated plants had higher LNA at all levels of treatment (0%N

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up to 5%N) compared to N + P treated plants, the rate of LNA increase was slower compared to the rate of increase observed in the N + P treated plants. The N + P treated plants had LNA values of 0.68 g m-2 at 1%N and 1.03 g m-2 at 5%N, while the N - P treated plants at 1%N had LNA of 1.01 g m-2 and at 5%N 1.17 g m-2.

The greater rate of increase in LNA shown by N + P treated plants over N - P treated plants indicates that at certain nutrient addition rate above 5%N, the N + P treated plants will surely increase in LNA more than their corresponding N - P treated plants. It seems therefore that the presence of P in N + P solution slows down the plant response to LNA at low N + P levels but enhances LNA respond at higher levels of N + P treatments. However, the differences obtained between the N + P and N - P treated plants were not significant (p > 0.05) and also no significant interaction between N and P treatment was observed.

Sunflower (Figure 3B). In treatment with the lowest nutrient level of 1%N there was a clear difference in LNA obtained between the N + P (1.02 g m-2) and N - P (1.45 g m-2). The N - P treated plants increased in LNA at all nutrients levels from 1%N up to 7%N more than the N + P treated plants. At 10%N level however an insignificant increase in LNA (3.08 g m-2) was exhibited by the N + P treated plant over the N - P plants (2.85 g m-2). The trend of increase in LNA exhibited by the N - P treated plants was observed to slowly decrease as nutrient addition rates increased while with the N + P treated plants an increasing trend with increasing N + P treatment levels was observed. The increasing trend exhibited by N + P treated plants plus the fact that at 10%N level N + P plants were a little higher in LNA more than their corresponding N - P treated plants counterparts, indicate that around N + P levels of 10%N and surely above that level, the N + P treated plants should be higher in LNA compared to N - P treated plants. It seems therefore that the presence of P in N + P solution slowed down plant respond to LNA at nutrient levels below 10%N but enhances plant responds to LNA at nutrient level of 10%N (similar result obtained in silver birch Figure 3A). The observed differences exhibited by N + P and N - P groups of plants were statistically revealed to be significance with P treatment (p < 0.05) but with no interaction between N and P.

Comparison of the two plant species revealed that increase in LNA was P insignificant in birch and P significant in sunflower. The N*P interaction was insignificant in both species. Only at nutrient level of 10%N did the N + P treated plants increase in LNA more than their corresponding N - P treated plants in the sunflower experiment, while in silver birch at nutrient level 5%N the N + P treated plants were higher in LNA than the N - P treated plants. These suggest therefore that the presence of P in N + P solution increases plant response to LNA in silver birch at lower concentration (5%N) compared to the sunflower (10%N) irrespective of the fact that P was statistically insignificant in silver birch and significant in sunflower. A possible reason thus, why at lower N + P levels (5%N) greater response in LNA was observed in birches than in sunflower (10%N) might be that the birches had a larger storage reserve of P compared to sunflower. At their common nutrient level of 5%N, however the sunflowers were higher in LNA (1.5 g m-2) than birches (1.2 g m-2).

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Leaf nitrogen per unit mass LNM (mg g-1) vs nutrient treatment

Leaf nitrogen per unit mass was observed to increase as the nutrient addition rates increased in both species and in both N + P and N - P treatments (Figure 3C, 3D). The increase was tested to be significant p < 0.05 with nitrogen application in both treatments and species.

Silver birch (Figure 3C). There was no difference in LNM obtained between P-treated (10.2 mg g-1) and P-deficient (10.1 mg g-1) treatment groups at 0%N levels. As nutrient levels were increased to 1%N the N - P treated plants increased more in LNM (18.9 mg g-1) than the N + P-treated plants (16.0 mg g-1). At 5%N the N + P treated plants increased by 1.0 mg g-1 more (31.4 mg g-1) than the N - P plants (30.4 mg g-1). Since at 1%N the N - P treated plants increased in LNM a little more than the N + P and at 5%N decreased in LNM than N + P treated plant, it seems therefore that increase in LNM exhibited by the N - P plants decreases with increasing N - P treatment levels, while that of N + P treated plants increases as N + P levels increases. The fact that at 1%N level the N + P treated plants were lower in LNM and higher in LNM at 5%N than the N - P treated plants, indicates that the presence of P in N + P solution at low treatment levels might had slowed down the response to LNM, and enhance the response at higher levels of 5%N and above. The differences obtained between N + P treated plants and N - P treated plants were shown to be statically significant (p = 0.02), but with no significant interaction (p = 0.29) between N*P.

Sunflower (Figure 3D) as the N - P treated plants exhibited higher LNM at all treatment levels more than the N + P treated plants, however the difference was pretty much obvious from 1%N up to 3%N. It seems therefore that the N + P treated plants were picking up the pace of increase as from nutrient concentration levels above 3%N. which explains the observed decrease in difference between the treatments (N - P and N + P) as treatments levels increases. Therefore the presence of P addition in N + P solution did not seem to influence changes LNM in sunflower as did in birch. The differences obtained between the N + P and N–P treatments were statistically shown to be significant (p = 0.02) with P and insignificant (p = 0.58) with N*P interaction.

Comparison of the two plant species gave that the effect of P was significant and the effect of N*P was insignificant in both species. The N + P treated birches at 5%N level had highest LNM while the sunflower even at 10%N level failed to accumulate LNM more than their N - P counterpart. At their common nutrient level of 5%N the birches accumulated a slightly higher LNM of (31.2 mg g-1) more than the sunflower (29.2 mg g-1).

Above ground nitrogen content AGN (mg) vs total nitrogen added

Above ground nitrogen increased with nutrient addition rates in both plant species and in both N + P and N - P treatments. This effect was statistically significant (p<0.05). (Figure 4A, 4B)

Silver birch (Figure 4A). There wasn’t a significant difference in AGN obtained between N + P treated plants and N - P treated plants at 0%N level. As nutrient levels increased to 1%N the N - P treated plants increased in AGN (110 mg) more than N + P treated plants (70 mg). By further increasing the nutrient level to 5%N, the N + P treated plants were observe to

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increase (230 mg) more than N - P treated plants (140 mg). This indicates that the presence of P at low nutrient levels of 1%N slowed down the rate of nitrogen uptake and accumulation, and enhances the uptake rate and accumulation at higher nutrient levels of 5%N. A total AGN of 330 mg was exhibited by the N + P treated plants compared to 270 mg exhibited by N - P treated plants. This implies that the rate of above ground nitrogen accumulation increases with increasing P concentration. The effect of P to AGN was statistically significant (p = 0.14), as well as the interactive effect of N and P (p = 0.01).

Sunflower (Figure 4B). At 1%N level, did N - P plants increased (6.1 mg) in AGN more than N + P treated plants (2.7 mg), at 2%N there was no significant difference observed. As from 3%N level the N + P treated plants began increasing more than the N - P plants, the increase was progressive with increasing P application levels. Thus the more P added the more difference in AGN obtained between N + P treated plants and N - P treated plants. The highest increase in AGN (66.9 mg) was obtained at the highest P application level of 10%N, at this 10%N level the N + P treated plant increased by (14.5 mg) more than N - P treated plants. The lowest increase (8.3 mg) was obtained at P application level of 3%N, at this nutrient level the N + P treated plant increased by (1.5 mg) more than N - P treated plants. Taking the total of all the N + P treated plants and N - P treated plants gave a total AGN of (123.12 mg) N + P treated and (103.28 mg) N - P treated plants. In sunflower therefore P application was observed to significantly enhance the rate of AGN accumulation. The effect of P to increase in AGN was statistically significant p = 0.04, while the interactive effect of N and P was insignificant p = 0.06.

Figure 5. Above ground dry mass (g) vs accumulated above ground nitrogen (g). In silver birch A, p-values are (N p=0.01, P p=0.96, N*P interaction p=0.06), the regression functions of +P (y = 24.275x + 5.8832, R² = 0.9441) and –P (y = 34.412x + 5.415, R² = 0.3898). And in sunflower B, p-values are (N p=<0.0001, P p=<0.0001, N*P interaction p=0.01), the regression functions of +P y = 24.985x + 0.9809 (R² = 0.7202) and –P y = 19.161x + 0.3843 (R² = 0.9248). Error bars indicate one standard error. +P and –P stands for plus phosphorus and without phosphorus respectively 

Comparison of the two species gave an insignificance of P in birch and P significance in sunflower to AGN increase. Also N*P was shown to be significant in birch and insignificant

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in sunflower. Increases in AGN commence with regard to P application around similar levels of P treatment (3% - 4%) in both species. This range of treatment levels therefore indicates the minimum N + P levels required to enhance the accumulation of above ground nitrogen.

Above ground dry mass (AGDM g) vs nitrogen content of aboveground biomass (AGN) (g)

Above ground dry mass (AGDM) was observed to increase significantly in both species and in N + P and N - P treated groups of plants. The effect of nitrogen to the increase in AGDM observed in both species was statistically tested to be significant p<0.05 (Figure 5A and 5B).

Silver birch (Figure 5A). Plants from N + P and N - P responded similarly to increasing amount of AGN, however, the responses of the N - P treated plants were more variable, shown by the AGDM vs AGN regressions for N + P treated plants (R² = 0.9441) and N - P treated plants (R² = 0.3898). The N + P treated plants had a total AGDM 25.7 g slightly above 25.5 g of N - P treated plants. Thus there was no significant difference in total AGDM obtained between the treatments. Nevertheless at 1%N, the N + P plants were lower in AGDM compared to N - P plants, indicating that the presence of P may have slowed down AGDM accumulation by slowing down the accumulation of AGN. At 5%N N + P plants were higher in AGDM compared to N - P plants, indicating that P may have enhanced the AGDM accumulation by enhancing the accumulation of AGN at same treatment level. This implies therefore that rate of increase in AGDM is dependent on the rate of increase AGN content, which in turn is dependent on the concentration of N and P in the nutrient solution. The effect of P to increase in AGDM was however statistically insignificant p=0.96, and the interactive effect of N and P was also insignificant p=0.06.

Sunflower (Figure 5B). AGDM was shown to increase with increasing AGN content. The plants treated with N + P solution increased in AGDM more than N - P treated plants. In Figure 4, the presence of P in N + P solution was shown to enhance AGN accumulation, a root cause of why the N + P plants accumulated higher AGN compared to N - P treated plants. The increase in AGDM exhibited by N + P plants over N - P plants is due to P effect in N + P solution. Regression lines with positive correlation were exhibited by both treatments, (R2 = 0.72) for N + P treated plants, and (R2 = 0.92) for N - P treated plants. The N + P plants showed a somewhat linear increase from 1%N to 5%N treatment levels and there after a bend was observed that progressed to 10%N. Indicating that at 5%N the N + P plants reached their optimal growth rate. At from 3%N up to 10%N treatment levels the N + P plant increased in AGDM about twice more than N - P treated plants, and total AGDM for N + P treated plants was about two fold higher (8.96g) higher than the N - P plants (4.28g), clearly indicating the role of P in enhancing AGDM accumulation. The effect of P to increase in AGDM was statistically tested to be significant p=<0.0001, as well as the interactive effect of N and P (p=0.01).

Comparison of the species gave an insignificant effect of P in silver birch and a significant P effect in sunflower on the AGDM accumulation. Addition of P seems to have a large positive effect on AGDM in sunflowers, while not in silver birch.

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Figure 6. Relationship between leaf mass per area (gm-2) A and B and Leaf thickness (mm) C and D: vs accumulated leaf nitrogen per area (gm-2). P-values for A (N p=0.0001, P p=0.0001, N*P interaction p=0.008), regression function for +P (y = 0.3968x - 0.1907, R² = 0.9154) and –P (y = 0.0771x + 0.0492, R² = 0.3978). P-values for B (N p=0.002, P p=0.03, N*P interaction p=0.79), regression functions for +P (y = -30.304x + 64.108, R² = 0.9997) and –P (y = -24.909x + 72.547, R² = 0.6298). P-values for C (N p=0.08, P p=0.78, N*P interaction p=0.18), regression functions (y = -12.327x + 87.496, R² = 0.3757) and –P (y = -5.2188x + 76.954, R² = 0.1264). P-values for E (N p=0.36, P p=0.59 and N*P interaction p=0.92), regression function for +P (y = 0.0075x + 0.1483, R² = 0.3409) and -P (y = 0.0178x + 0.1403, R² = 0.935) and P-value for D (N p=0.73, P p=56 and N*P interaction p=0.41), regression functions for +P (y = -0.0084x + 0.2719, R² = 0.2231) and –P (y = 0.0143x + 0.2198, R² = 0.2361). +P and –P stands for nutrient including phosphorus and no phosphorus respectively. Trend lines are linear regression. Error bars indicate standard deviations from the mean.

Leaf mass per area (LMA gm-2) vs leaf nitrogen per area (gm-2)

Leaf dry mass per unit area (gm-2) was negatively correlated with leaf nitrogen per unit area. A decreasing trend in LMA with increasing LNA content was observed (Figure 6A. 6B). This affirms that nutrient treatment did not actually have a positive effect on biomass allocation to foliage in per area basis in both plant species when examined in relation to leaf nitrogen per area basis.

In silver birch (Figure 6A), higher LMA contents were observed in N - P plants than N + P treated plants, suggesting that P treatment may have reduced the ability of the plants to accumulate LMA content. The highest increase in LMA in both N + P treated and N - P treated plants were observed at 0%N level while the lowest was at 5%N level. The differences in LMA responses observed between N + P and N - P treated plants were statistically shown to be significant with N p=0.002, P p=0.03, and insignificant with N*P interaction p=0.79.

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In the sunflower (Figure 6B), there was not so great difference in LMA observed between N + P treated plants and N - P treated plants. However as leaf nitrogen per unit area increased, a fall in LMA was observed in N + P and N - P treatments although the decrease was a little more obvious in N + P plants. The regression line of N + P plants was observed to decrease slightly more than in N - P plants. The differences in LMA observed between N + P and N - P plants were statistically insignificant with N p=0.08, P p=0.78 and N*P interaction p=0.18. Hence changes in LMA with respect to leaf nitrogen per unit area were more expressed in silver birch than sunflower, and the effect of Phosphorus to these changes was significant in silver birch and insignificant in sunflower.

Nevertheless when leaf dry mass (LDM) was examined in relation to leaf nitrogen content other than LNA a positive correlation was observed at all levels of treatments in both species. LDM was observed to increase as leaf nitrogen content increased; the increase was however observed to be higher in N + P plants than N - P plants (Table 3). The lowest and highest LDM for birch (0.09 g) and (0.21) was obtained at 0%N and 5%N, and in sunflower (0.04 g) and (0.17 g) obtained at 1%N and 10%N respectively (Table 3). The LDM was observed to increase progressively up to at 5%N in both birch and sunflower. However above 5%N levels in sunflower rate of increase in LDM began to slowly decrease (see Table 3), this implies that at 5%N the sunflower plants may have possibly reached their optimum growth rate.

Table 3. Mean values and standard deviations (StdDev) of leaf thickness (LT), leaf area (LA) and leaf dry mass (LDM) of silver birch and sunflower leaves harvested after gas exchange measurements. Data shows increase in LA and LDM as fertilization levels increases; there aren’t great changes in leaf thickness observed with respect to increase levels of nutrient application with species. The sunflowers are higher in LT, LA and LDM compared to birches. The N + P treated plants were higher in LA and LDM than the N - P treated plants in both birches and sunflowers

Species  Treatment  Mean Leaf Thickness (LT mm) 

StdDev  of  LT  

Mean  Leaf  Area  (LA cm2) 

StdDev 0f LA 

Mean Leaf Dry mass (LDM g) 

StdDev Of LDM 

Silver birch  0%N + P  0.15  0.01  19.2  4.1  0.09  0.02 

Silver birch  1%N + P  0.16  0.01  30.7  9.9  0.13  0.03 

Silver birch  5%N + P  0.16  0.01  33.1  0.4  0.11  0.01 

Silver birch  0%N – P  0.15  0.01  15.8  0.0  0.09  0.03 

Silver birch  1%N – P  0.16  0.02  23.9  5.0  0.13  0.01 

Silver birch  5%N – P  0.16  0.01  54.6  8.5  0.21  0.02 

             Sunflower  1%N + P  0.27  0.02  4.2  0.6  0.04  0.01 

Sunflower  2%N + P  0.25  0.01  6.0  1.3  0.05  0.01 

Sunflower  3%N + P  0.27  0.02  10.9  1.7  0.07  0.01 

Sunflower  5%N + P  0.24  0.01  14.8  2.1  0.08  0.02 

Sunflower  7%N + P  0.27  0.08  14.0  2.6  0.07  0.02 

Sunflower  10%N + P  0.24  0.05  30.6  15.4  0.17  0.06 

Sunflower  1%N – P  0.24  0.01  6.3  2.1  0.04  0.01 

Sunflower  2%N – P  0.25  0.01  5.2  2.2  0.04  0.02 

Sunflower  3%N – P  0.23  0.01  3.7  2.0  0.03  0.01 

Sunflower  5%N – P  0.27  0.02  6.8  1.4  0.05  0.03 

Sunflower  7%N – P  0.27  0.02  8.2  3.3  0.06  0.05 

Sunflower  10%N – P  0.25  0.04  13.7  6.5  0.07  0.03 

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Leaf Thickness (mm) vs leaf nitrogen per unit area (gm-2)

Increasing levels of LNA didn’t significantly (p>0.05) affect leaf thickness (LT) in both birch (Figure 6C) and sunflower (Figure 6D). There was no clear thickness difference observed between N + P treated plants and N - P treated plants in both species. Nevertheless the sunflowers had thicker leaves than birches at their common nutrient treatment levels. The regression correlation was somewhat horizontal in both species and treatments. The effect of N, P and N*P to change in LT in both species and treatments were statistically insignificant p>0.05.

Maximum rate of carboxylation (Vcmax) on leaf nitrogen per area basis

Silver birch (Figure 7A), Vcmax was observed to increase with increasing levels of LNA content in both N + P and N - P treated plants. Although the LNA content of N - P plants were higher than the N + P plants at all levels of treatment, there was no significant difference in Vcmax observed at 0%N and 1%N treatments levels between N + P and N - P treated plants. At 5%N treatment level the N + P plants were higher in Vcmax (52.3 μmol m-2 s-1) than the N - P treated plants (45.0 μmol m-2 s-1). Thus the N + P treated plants were (12.0 μmol m-2 s-1) higher in Vcmax over the N - P plants. However, no significant effect of P (p=0.54) or N*P (p=0.6) was observed on rate of carboxylation, but the the effect of N (p=<0.0001) was highly significant.

Sunflower (Figure 7B) a progressive increase in Vcmax with increasing LNA content between 1 - 1.5 g N m-2 was exhibited by the N + P plants, while above N m-2 a decrease in Vcmax was observed. The Vcmax of the N - P plants had no correlation with LNA. The highest Vcmax (116.9 μmol m-2 s-1) was exhibited by N + P treated plants at 5%N treatment level. The N + P plants were therefore (17.9 μmol m-2 s-1) Vcmax greater over the N - P plants. However, no significant effect of P (p=0.14) was observed while there was a significant interaction N*P (p=0.01).

Comparison of the two plants species revealed that although in both cases the effect of P was statistically insignificant, the presence of P in N + P solution however increased the rate of carboxylation in both species. The highest Vcmax rates obtained in both species were at 5%N nutrient treatment levels. Implying that at 5%N treatment was sufficient to fulfil the demand of N to reached their maximum carboxylation rates.

Maximum rate of electron transport (Jmax) vs leaf nitrogen per unit area

Silver birch (Figure 7C) Jmax was observed to increase as LNA content increased in both treatments. At 0%N and 1%N treatment levels there was no significant difference in Jmax observed between N + P and N - P treated plants. However at 5%N treatment level the N + P treated plants increased in Jmax by (25.5 μmol m-2 s-1) more than N - P plants. This implies therefore that P has no effect in Jmax at low concentration, but increases the rate of electron transport at certain high concentrations.. A difference of 68.4 μmol m-2 s-1 was exhibited by N + P treated plants over the N - P plants. These differences were statistically significant with N (p=<0.0001), insignificant with P (p=0.17) and N*P interaction (p=0.09).

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Sunflower (Figure 7D) Jmax was observed in N + P treated plants to increase progressively with increasing LNA contents. The N - P plants were observed to fall in Jmax as LNA content increased. The highest Jmax rate of (167.3 μmol m-2 s-1) for N + P plants was obtained at 10%N, while that of N - P plants (90.4 μmol m-2 s-1) was obtained at 1%N level. This indicates that P enhances the rate of electron transport in sunflower plants.

The differences observe between the N + P and N - P treatments were statistically significant with N (p=0.0005) and P (p=<0.0001) application as well as N*P interaction (p=<0.0001).

In both plant species therefore P application was observed to increase the rate of electron transport (Jmax). The increase was proportional to the amount of P added. In the birch the increase in Jmax was obtained at 5%N level while in sunflower an increase was initiated from 1%N level. This indicates therefore that the sunflowers utilize P better than the birches and that the rate of electron transport is higher in sunflower than in silver birch.

Figure 7. Relationship between Vcmax (μmol m-2s1) A and B, Jmax (μmol m-2 s-1) C and D, Vs leaf nitrogen per area (LNA g m-2). P-values for A (N p=0.0004, P p=0.5, N*P p=0.6) regression function for +P (y = 55.594x - 3.6554, R² = 0.9529) and –P (y = 46.824x - 8.3702, R² = 0.9822). P-values for B (N p=0.31, P p=0.14, N*P p=0.01) regression functions for +P (y = 22.555x + 49.766, R² = 0.3232) and –P (y = -11.354x + 99.414, R² = 0.1768). P-values for C (N p=<0.0001, P p=0.17, N*P p=0.09) regression functions for +P (y = 96.288x - 2.2207, R² = 0.9383) and –P (y = 48.13x + 16.939, R² = 0.8805). P-values for D (N p=0.0005, P p=<0.0001, N*P p=<0.0001) regression functions for +P(y = 38.652x + 59.371, R² = 0.5538) and –P (y = -2.1022x + 69.973, R² = 0.0036). +P and –P stands for nutrient including phosphorus and little/no phosphorus respectively. Trend lines are polynomial regression. Error bars indicate standard deviation from the mean.

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Quantum yield vs Leaf nitrogen per unit area (gm-2)

Measuring net CO2 fixation in intact leaves at fixed CO2 concentration of 400 mmol mol-1 and varying photon fluxes gave way to construct light response curves that provided useful information about the photosynthetic properties of the leaves of both silver birch and sunflower. Information extracted from the light curves was quantum yield (Φ ) (Figure 8) and mitochondria respiration (Rd) (Figure 9). Quantum yield readings were taken at light intensities bellow kok effect and at light intensities above kok effect. In both the birch and sunflower and both N + P treated and N - P treated plants, Quantum yield at light intensities (bellow Kok effect) was observed to be about twice higher than at light intensities (above Kok effect). In birches at low PPFDs below the Kok effect, Quantum yield Φ was observed to increase from 0%N to 1%N and then decrease as LNA content increased in both N + P and N - P treated plants (Figure 8A). Phosphorus application was not observed to enhance the rate of CO2 fixation per intercepted photon as the effects (N p=0.26, P p=0.88 and N*P p=0.51) were all statistically insignificant. The highest quantum yields were obtained at 1%N, (0.073 mol mol-1) for N + P treated plants and (0.066 mol mol-1) for N - P plants (Figure 8A), while above the Kok effect (Figure 8C) quantum yields at the same 1%N treatment level were observed to drop by half (0.044 mol mol-1 for N + P plants and 0.034 mol mol-1 for N - P plants). Generally, quantum yield Φ above the Kok effects were observed to increase as LNA content increased in N + P and N - P treatments (Figure 8C). There was a positive correlation (R² = 0.8057) observed between Φ and LNA. The highest rates of Φ were at 5%N in both N + P treated (0.05 mol mol-1) and N - P (0.04 mol mol-1) plants. Phosphorus application was not observed to significantly enhance the rate of CO2 fixation per photon intercepted. The effect of N (p=0.07) was almost statistically significant while that of P (p=0.41) and N*P interaction (p=0.46) were clearly insignificant. In sunflowers below Kok effect, the N + P plants were observed to increase while the N - P plants were somewhat stable in quantum yields as LNA contents were increasing (Figure 8B). The highest quantum yield for N + P plants was (0.08 mol mol-1) at 10%N level and for N - P plants (0.06 mol mol-1) at 3%N level (Figure 8B). Phosphorus in N + P solution therefore seems to have enhanced the rate of CO2 fixation per photon absorbed in N + P treated plants. The effect of P (p=<0.0001) was statistically significant, while that of N (p=0.06) and N*P interaction (p=0.17) were insignificant. Above the Kok effect (Figure 8D), quantum yields were observed to drop by half compared to quantum yield below Kok effect. The N + P treated plants were observed to increase in Φ from 1%N up to 5%N treatment levels; above 5%N level a decrease in Φ was obtained. The N - P treated plants were observed to decrease in Φwith increasing LNA content. Phosphorus seems to influence the rate of quantum yield in N + P treated plants. The effects of N was

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statistically insignificant (p=0.11), while that of P (p=<0.0001) and N*P interaction (p=0.0002) were significant. w2

Figure 8. Effect of leaf nitrogen in per unit area basis on photosynthetic quantum yield, A and B shows the quantum yield at low light intensities up to Kok effect (were the linear responds to photosynthesis at low light intensities begins to bend off) and C and D show quantum yield at light intensities above the Kok effect. P-values for A (N p=0.26, P p=0.88, N*P p=0.51) regression functions for +P (y = -0.1192x2 + 0.1827x, R² = 0.7475) and –P (y = -0.1119x2 + 0.1795x, R² = 0.9985). P-values for B (N p=0.06, P p=<0.0001, N*P p=0.17) regression functions for +P (y = -0.0145x2 + 0.0722x, R² = -0.445) and -P (y = -0.0115x2 + 0.0483x, R² = -0.151). P-values for C (N p=0.07, P p=0.41, N*P p=0.46) regression function for +P (y = -0.0376x2 + 0.0851x, R² = 0.8057) and –P (y = -0.0334x2 + 0.0728x, R² = 0.3376). P-values for D (N p=0.11, P p=<0.0001, N*P p=0.0002) regression function for +P (y = -0.0106x2 + 0.0435x, R² = -0.217) and –P (y = -0.0058x2 + 0.025x, R² = -0.223). +P and –P stands for nutrient including phosphorus and no/low phosphorus respectively. Trend lines are polynomial regression. Error bars indicate one standard error.

Mitochondria respiration (Rd) vs Leaf nitrogen per unit area

Mitochondria respiration as a function of leaf nitrogen per unit area was generally observed to increase in both species and treatments (Figure 9). However the N + P treated plants were higher in Rd than the N - P plants in both species. This implies that Phosphorus application did equally enhance the plants response to Rd. Just like in quantum yield Rd was almost twice higher bellow Kok effect compared to above the Kok effect. This indicates therefore that the

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rate of Φ is somehow dependent on the rate of Rd, implying that quantum yields are higher bellow the Kok effect as a result of the higher Rd observed bellow Kok effects. ANOVA test revealed the differences observed between N + P treated and N - P plants to be for (Figure 9A) N p=0.005, P p=0.13, N*P p=0.18, (Figure 9B) N p=0.03, P p=0.003, N*P p=0.05, (Figure 9C) N p=0.009, P p=0.05, N*P p=0.3 and (Figure 9D) N p=0.6, P p=0.3, N*P p=0.14.

Figure 9. Nutrient (Leaf nitrogen/area gm-2) effect on rates of Mitochondria respiration (Rd) in birch and sunflower at low PPFD up till Kok effect Figure A and B, and at PPFD above Kok effect Figure C and D.P-values for A (N p=0.005, P p=0.13, N*P p=0.18) regression functions for +p (y = 1.5007x + 0.2657, R² = 0.5046) and –P (y = 3.5908x - 2.7673, R² = 0.8394). P-values for B (N p=0.03, P p=0.003, N*P p=0.05) regression functions for +P (y = 0.6157x + 1.0214, R² = 0.5983) and –P (y = 0.1088x + 1.2534, R² = 0.0318). P-values for C (N p=0.009, P p=0.05, N*P p=0.3) regression functions for +P (y = 1.9174x - 0.7611, R² = 0.8024) and –P (y = 0.7891x - 0.4723, R² = 0.7924). P-values for D (N p=0.6, P p=0.3, N*P p=0.14) regression functions for +P (y = 0.1016x + 0.9774, R² = 0.0331) and –P (y = 0.1315x + 0.6646, R² = 0.1385). 

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Leaf Photosynthetic capacity (Amax) as a function of nitrogen added in mg

In both birches and sunflower the N + P plants were observed to increase in Amax with increasing treatment levels (Figure 10).

In birches the increase was from 0%N all the way to 5%N (Figure 10A), and in sunflower the increase was observed from 1%N up to 5%N level, and above 5%N to 10%N level a fall in Amax was observed (Figure 10B). The fall in Amax from 5%N level may imply that above this treatment level, N + P application in sunflower maybe excessive to the plants. Thus the nutrient concentration level required to obtain maximum photosynthetic capacity in sunflowers is 5%N. In the birch (Figure 9A) it wasn’t possible to reach the N + P nutrient concentration level required for maximum Amax because of the small number of replicas and nutrient levels used. If the nutrient levels used in birch were as in sunflower, reaching the N + P requirement for maximum Amax could have been possible. Nevertheless an increasing trend of Amax with increasing concentration levels of N + P treatment was observed up to at 5%N level, implying that N + P nutrient treatment levels of above 5%N are required to reach maximum photosynthetic capacity.

In both plants species the N - P plants were observed to decrease in Amax as nutrient levels increased. In the birches an increase in Amax was observed from 0%N to 1%N treatment level thereafter a fall in Amax was observed (Figure 10A). In the sunflower a decreasing trend in Amax was observed from 1%N to 3%N level, at 5%N a rise in Amax was observe and thereafter a fall was observed (Figure 10B). In both species at all levels of treatments except at (0%N birch) and (1%N sunflower) the N + P treated plants were higher in Amax than the N - P treated plants. The difference between N + P and N - P treatments were statistically shown to be (N p = <0.0001, P p = 0.12, N + P p = 0.14) for the Birches and (N p = 0.53, P p = 0.005, N + P p = 0.06) for the sunflower.

In both birches and sunflower therefore P application was observed to enhance leaf photosynthetic capacity and thus plant growth, but the enhancement was more expressed in sunflower than in birch, a possible reason for the significance of P in sunflower and its insignificance in birch.

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Figure 10. Leaf photosynthetic capacity (Amax) as a function of nutrient ttreatment examined in A birch and in B sunflower. P-values for A (N p=<0.0001, P p=0.12, N*P p=0.14) and for B (N p=0.53, P p=0.005, N*P p=0.06). The +P in both A and B increases in Amax as nutrient levels were increased up to at 5%N. +P and –P stands for nutrient including phosphorus and nutrient with no/low phosphorus respectively. Error bars are standard deviations from means.

DISCUSSION:

Accumulated Nitrogen per unit leaf area and mass basis

Examining the nitrogen content of the leaves per unit area and mass gave an increase in leaf nitrogen content with increasing nutrition addition in both birch and sunflower (Figure 3). The relative increase in leaf NN - P plants increased a little more in leaf N content compared to N + P treated plants. An explanation of this difference comes from the induced effect of P application on plants supplied with N + P. The N + P treated plants were observed to increase in leaf surface area more than the N - P plants; a similar result was obtained by Fredeen et al (1989) who observed a decrease in soybean leaf area at low P application. The number of mesophyll cells per unit leaf area and mass decreases progressively with increasing leaf size (Sasahara 1981), the increase in mesophyll cell number per unit leaf area with decreasing life size correlates with an increase in nitrogen content and increase in photosynthetic capacity (Sasahara 1981). Therefore in spite of the greater leaf surface area exhibited by the N + P treated plants, it appears that increase in mesophyll cell number per unit leaf area and mass enables a high rate of photosynthesis in N - P treated plants. This explains the higher N content observed in N - P treated plants at basis LNA and LNM. When changes in above ground nitrogen content (AGN) (Figure 4) in relation to total nitrogen added were examined, the N + P treated plants were observed to accumulate more N than N - P treated plants in both plant species. Shoot growth was observed to significantly decrease in N - P treated plants compared to N + P treated plants; a similar result was obtained by Fredeen et al (1989) on soybean plants. The decrease shoot growth was as a

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result of smaller leaf size and decrease in total leaf area, a consequence of total low N content, low rate of growth and photosynthesis. Which resulted in a decrease in AGN for N - P treated plants compared to N + P treated plants. At common nitrogen (N + P) addition of 5%N, the N content per unit leaf area had 40% higher LNA in sunflowers compared to birches. There wasn’t any significant difference observed when leaf nitrogen per unit mass was considered. However, the AGN was much higher in the birches (77 mg N per plant) compared to the sunflowers (5.7 mg N per plant) This maybe because the birch plants may have accumulated and stored N in their storage tissues developed before the start of the treatments as they were started from half meter tall seedlings while the sunflowers started from seed. The increase in LNA in sunflower compared to birch may imply that at a common Nutrient (N + P) level of 5%N, the sunflower take up and utilizes P more efficiently than the birch. Its however possible that this difference is due to the differences in light intensities used in the growth chambers when growing the plants. The sunflowers were grown at light intensities of (665 μmol m-2s-1) and birch at (60 μmol m-2 s-1). According to Evans (1989), plants grown in stronger light will have more N partitioned to soluble proteins. Since the increase in soluble protein is associated with increased RuBisCo activity (Evans 1989) the stromal nitrogen content will increase in respond to increase uptake of N added. Change in Biomass

The leaf mass per unit area (LMA) was observed to decrease in both species as leaf nitrogen content per unit area was increased (Figure 6A, 6B). This result is similar to that of Makino et al (2009) who reported that photosynthetic nitrogen use efficiency of tree leaves decreases with increasing LMA towards decreasing N and P availability. The decrease in LMA was more pronounced in the N + P treated plants of both species, but the difference between N + P and N - P treated plants was about twice in birches compared to sunflowers. At their common nutrient level of 5%N the LMA in sunflowers was about twice higher than that of birches, implying that sunflowers may be more efficient in taking up and utilizing P than birches. The possible explanation of the decrease in LMA with increasing LNA may come from the findings of Makino et al (2009) who reported that increases in leaf nitrogen content per unit area, increases N partitioning to the cell walls at the expense of photosynthetic enzymes. The consequence of this is reducing photosynthetic capacity (Amax), brought about by decrease amount of photosynthetic enzymes and reduce mesophyll conductance. In this study there was no significant change in leaf thickness when nutrient levels were increased in both species. Increases in cell walls are expected to increase leaf thickness (Terashima et al .2005), but this wasn’t the case in this study. Nevertheless there was an increase in leaf area with increasing nutrient treatments in both species. Therefore although Amax seems to decrease with LMA, when the entire leaf area was accounted for, the photosynthetic capacity was observed to increase (Figure 10). This was in line with the increase in above ground dry mass (AGDM) exhibited by N + P treated sunflower plants (Figure 5) when AGDM was examined in respond to above ground nitrogen (AGN). This is in accordance with the report of Uri et al (2003) who obtained and

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increase in above ground biomass with increase P and potassium treatments in Grey Alder (Alnus incana, (L.) Moench). Should above ground biomass be examined in per unit area basis, the N - P plants will accumulate higher AGDM compared to N + P treated plants, as a result of decrease shoot growth, increase numbers of mesophyll cells per unit leaf area and increase starch concentration in cells (Fredeen et al 1989). When AGDM was examined in respond to AGN (Figure 5) the total AGDM for N + P and N – P treated plants were both 26 g. A large difference though was observed in sunflower; the N + P treated plants were about twice higher in total AGDM than the N - P plants. The sunflower result correlated well with that of Brahim et al (1996) who reported a dry weight of shoots and roots increase of pine seedlings with increasing P supply. A possible explanation of the insignificant difference observed in birch is that the wood had some amount of P stored in their storage reserves, which was being transferred to the metabolic pool to maintain growth and photosynthesis. Thus external supplies were not taken up to the same extent. This is difficult to evaluate since P analysis was not performed in this study.

Vcmax and Jmax on leaf N per unit area basis

The photosynthetic parameters Vcmax and Jmax in relation to leaf nitrogen per unit area were observed to increase with increasing N + P application in both species (Figure 7). Similar to the finding by Bowen et al (2006) both Vcmax and Jmax increased strongly with increasing N and P supply in both species (Figure 7). However, in the sunflower experiment, the response at the higher N contents level was decreasing (Figure 7B, 7D) indicating a surplus in N supply. These results is in accordance with Romero et al (1999) who obtained an increase in Vcmax on Phragmites australis when N and P were gradually increased and a decrease in Vcmax at very high N and P application levels. Bown et al (2006) also studied the effect of N and P separately by keeping N in one case limited and P in the other limited. Their result showed an increase in Vcmax and Jmax when with increasing concentration of both N and P.. This result is in line with that observed in this study on birches but not on sunflowers. When P was limiting, P deficient birch plants still increased in Vcmax and Jmax (Figure 7A, 7C) while in sunflower a decrease in Vcmax and Jmax was observed (Figure 7B, 7D). Phosphorus has been reported to interact with the N metabolism of plants (Reich et al 2009). P is important in plants for the formation of nitrate-reducing enzymes (Reich et al 2009) as well as formation of RuBP in the Calvin cycle (Farquhar et al 1980, Reich et al 2009), therefore in the absence of P, the rate of protein synthesis will fall, meaning reduce amount of RuBisCO available to drive photosynthesis. In this case Vcmax and Jmax are expected to decrease, therefore explains the decrease in Vcmax (Figure 7B) and Jmax (Figure 7D) exhibited by N - P sunflower plants. A possible explanation therefore of the increase in Vcmax and Jmax exhibited by N - P birch plants and that observed by Romero et al (1999) in Phragmites australis is that as woody plants they both had some amount of P in their storage reserves which were being transferred to the metabolic pool when P was limiting to maintain growth and photosynthesis.

Quantum yield and Mitochondria respiration (Rd)

Quantum yield (the ratio of CO2 assimilated to the number of absorbed photon) obtained from the light curves was observed to increase with increasing nutrient treatment levels in

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both species. The increase in quantum yield was more exhibited by the N + P treated plants compared to N - P plants in both species (Figure 8). In sunflower experiment, the quantum yield in the N - P plants was observed to be negatively correlated with increasing LNA levels (Figure 8B, 8D). According to Lin et al (2009), phosphorus functions in regulating the electron transport processes by providing the orthophosphate (Pi) for ATP synthesis. The difference in quantum yield between N + P and N – P therefore can be attributed to the difference in amount of ATP synthesis. Increase amount of ATP will increase the rate of regeneration of RuBP, CO2 assimilation and hence quantum yield. A possible explanation of the increase in quantum yield observed in N - P birches (Figure 8C) however maybe as a result of the stored P in their storage reserves, which in the absent of P should be transferred to the metabolic pool to maintain growth and photosynthesis. Quantum yield was observed to decrease with increasing light intensities. A similar result was reported by Sharp et al (1984) in sunflowers. The decrease in quantum yield observed when light intensities were increased, according to Sharp et al (1984) was due to decrease in mitochondria respiration and increase in photorespiration with increasing light intensities. Mitochondria respiration (Rd) was examined at low light (bellow) (Figure 9A, 9B), and at light (above) (Figure 9C. 9D) the Kok effect (the point at which the linear response to photosynthesis at low light intensities begins to bend off) usually at the vicinity of the light compensation point. According to Sharp el al (1984), no CO2 assimilation takes place in the dark, instead O2 is taken-up and CO2 release through mitochondria respiration, as light intensities increases CO2 assimilation linearly increases until it equal CO2 release by mitochondria respiration the point known as Kok effect. Further increase in light above the Kok effect increases the Rubisco-Oxygenase affinity to O2 (photorespiration) with a consequence of decreasing CO2 assimilation rate, and hence quantum yield (Sharp et al .1984, Mebrahtu et al 1991). These therefore explains the high quantum yields observed at light bellow Kok effect compared to light above Kok effect in both plants species. Nevertheless the N + P treated plants were higher in Rd compared to N - P plants in both plant species (Figure 9), this correlates well with the findings of Theodorou et al (1993) who observed an increase rate of O2 uptake and CO2 release in Catharanthus roseus cells grown in P sufficient against P deficient mediums. The greater quantum yield exhibited by N + P treated plants over N - P plants is because Phosphorus functions in regulating mitochondria respiration by providing the orthophosphate (Pi) for ATP synthesis which provides the energy to runs the process (Theodorou et al. 1993). The birch plants treated with N + P solutions increased in quantum yield, particularly in (Figure 8C) were the increase was from 0%N to 5%N levels, while the sunflower treated with N + P solution increased in quantum yield linearly from 1%N up to 5%N treatment levels and then begins to bend off (Figure 8B, 8D). Therefore N + P nutrient treatment levels of 5%N are required to obtain optimal quantum yield, growth and photosynthesis in sunflower plants. It was not possible to reach the optimal nutrient requirement for optimum quantum yield in birch because of small number of replica and nutrient levels used, however the N + P treatment level required to obtain optimal quantum yield, growth and photosynthesis in

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birches should be above 5%N level, since at this level the regression line was still linearly increasing. Leaf photosynthetic capacity (Amax)

Leaf photosynthetic capacity (Amax) was observed to increase in both species as N + P applications were increased from 0%N up to 5%N level (Figure 10), above 5%N level Amax in sunflower was observed to decrease (Figure 10B). This result is in line with that obtained when AGDM, Vcmax, Jmax and quantum yield were examined. The N + P treated plants exhibited higher Amax than N - P plants in both species (Figure 10). A similar result was obtained by Brahim et al (1996) who observed a decrease in Amax in P-deficient Eucalyptus seedlings. The lower Amax observed in the N - P plants compared to the N + P plants can be related to a decrease in rate of RuBP regeneration (Brahim et al .1996, Romero et al 1999, Lin et al 2009) smaller amount of RuBisCO, and decrease in specific activity of RuBisCO (Brahim et al 1996) brought about by P-deficiency or P limitation. At a common N + P nutrient level of 5%N, the sunflowers were about twice higher in Amax than birches. P application therefore was observed to influence Amax in sunflowers more than in birches.

CONCLUSION:

The objective of this study was to verify the effect of N and P on photosynthesis and growth of silver birch and Sunflower. The results obtained revealed that nitrogen and phosphorus supply affected growth and photosynthesis at all levels of application. Nitrogen and phosphorus application significantly enhanced growth properties (leaf area, leaf dry mass, leaf fresh mass, leaf nitrogen content) and photosynthetic parameters (Vcmax, Jmax, quantum yield and Amax). The relationship between growth and photosynthesis with N and P addition was positively correlated. External nutrient additions were done using relative addition techniques, which involve the supply of nutrients in relation to plant growth rate. Leaf photosynthetic capacity (Amax) increased linearly with the amount of N and P taken up by the plants as was Vcmax, Jmax and quantum yield. In the absence or limiting amount of P Vcmax, Jmax, quantum yield and Amax in birches and sunflowers were all observed to decrease; a decrease brought about by low amounts of cellular orthophosphate (Pi), needed to build ATP and NADPH during photophosphorylation in the light reaction phase of photosynthesis, a consequence of decreased rate of regeneration of RuBP in the Calvin cycle. In the absence or at limiting levels of P therefore growth and photosynthesis will decrease as a result of reduced carboxylation rate. Hence for maximum growth and photosynthesis to be attained optimal levels of both N and P are required. In this study the N + P requirement for maximum photosynthesis was obtained at 5%N for sunflowers. For the birches the small number of treatment levels and replicates were not sufficient to reach the nutrient concentration required for optimal growth. However the concentration of N + P required for maximum photosynthesis in birch should be above 5%N, since at this concentration the regression line was still linearly increasing. At all levels of treatments plants supplied with N + P accumulated higher leaf-N content compared to N - P plants; thus P influenced the rate of N uptake and use efficiency in both birch and sunflower. Leaf photosynthesis capacity (Amax)

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and leaf-N content were higher in sunflower than birch when examined at their common N supply of 5%N; therefore the photosynthetic response to N + P supply was greater in sunflowers compared to the birches.

Acknowledgment:

I would like to thank and express my gratitude to my supervisor Dr. Göran Wallin for his immense support and assistance. His comments and remarks through the learning process of this research were very helpful. I will also want to thank Dr. Johan Uddling Fredin for helping out with very useful discussions. Furthermore I want to thank Mats Räntfors for his assistance in running nitrogen analysis and for helping out with useful discussions throughout the entire study process.

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